Staphylococcus aureus is the single most common cause of osteomyelitis in humans. Incidences of osteomyelitis caused by S. aureus have increased dramatically in recent years, in part, due to the appearance of community-acquired antibiotic resistant strains of this pathogen. The recent identification of S. aureus isolates with reduced vancomycin susceptibility indicates the possibility of a pathogen resistant to all current forms of therapy. Thus, the increasing prevalence of S. aureus associated bone infections make understanding the pathogenesis of this organism imperative. Recently, we have described the surprising ability of bone-forming osteoblasts to secrete the key inflammatory cytokine, IL-12, when exposed to S. aureus. This finding is particularly significant given the central role IL-12 plays in the induction of cell-mediated immune responses. Such immune responses are essential for the successful elimination of intracellular pathogens such as S. aureus. The ability of these cells to produce IL-12 points to a previously unrecognized role for osteoblasts in the generation of protective immune responses and the resolution of infection of bone. In this application we will determine whether the production of IL-12 seen in vitro is reproducible in both an in situ-like organ cultures of neonatal mouse calvaria and in vivo using an animal model developed in our laboratory. Furthermore, we will establish the clinical relevancy of these findings using bone sections from patients with S. aureus osteomyelitis. Finally, we propose to investigate the mechanisms responsible for inducing the production of this important cytokine by osteoblasts. Specifically, we will determine whether the expression of bacterial recognition Toll- like receptors underlie the ability of bacteria to induce IL-12 production in cultured osteoblasts. These studies are anticipated to demonstrate that bone-forming osteoblasts respond to S. aureus by the production of this pivotal inflammatory molecule, thereby expanding the recognized role of these cells to include being key components in host responses during bone infection.